JP2000344768A - New compound a-77543 and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new compound having excellent antitumor activity and antiviral activity and useful as an antitumor agent or an antiviral agent. SOLUTION: This new compound is expressed by the formula and is obtained by culturing a microorganism [e.g. Nocardia.sp-SANK 62,965(FERM BP-6,179) strain, etc.], capable of producing the compound of the formula and belonging to the genus Nocardia and by recovering the compound of the formula from the culture. When using the compound as an antitumor agent or an antiviral agent, its administration form is e.g. a tablet, a capsule, powder, granule, syrup or the like which is orally administered or an injection, etc., and its quantity administered usually is 1-100 mg/day which is taken once a day or as a few doses a day.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel compound useful as an antitumor or antiviral agent, a method for producing the compound, and an antitumor or antiviral agent containing the compound as an active ingredient.

[0002]

BACKGROUND ART As a compound structurally similar to the compound of the present invention, for example, mycobactin is known. Mycobactin is a siderophor
It belongs to the substance group called e). Siderophores are iron chelators produced by microorganisms and are highly produced in an iron-deficient environment, and are therefore considered to have a function of capturing iron and assisting microbial cells to take up. Siderophores typically have multiple, often three, hydroxamic acids or catechols as functional groups for chelating iron.

Mycobactin is distinguished from other siderophores by two chemical structural features: One is hydroxamic acid as a functional group for chelating iron.
And one phenolic hydroxyl group. Another characteristic is that it has a long fatty side chain having about 9 to 19 carbon atoms, so that many other siderophores are water-soluble, but mycobactin is fat-soluble. It is. The compound of the present invention also has these two characteristics and is considered to be a kind of mycobactin.

[0004] Snow wrote a review in 1970, classifying mycobactin into two types, P-type and M-type (Bacteriological Reviews 34, 99-125, 19).
70 years). That is, mild hydrolysis of mycobactin under alkaline conditions results in mycobactic acid (mycobactic acid).
Two partial structures are obtained, called acid and cobactin. Of these, those having a long fatty side chain in the mycobactin acid portion are P-type mycobactin, and those having a long fat side chain in the cobactin portion are M-type mycobactin (according to this classification method, the compound of the present invention Belongs to M type mycobactin). Snow also lists two mycobactins, M and N, as M-type mycobactins. Mycobactins M and N are names given to a series of homologues having different chain lengths of long fatty side chains, respectively, and both have different structures of the mycobactinic acid moiety from the compounds of the present invention. That is, the structure of the acyl group in the hydroxamic acid of the mycobactin acid moiety is such that mycobactin M is acetyl and mycobactin N is propionyl, whereas the compound of the present invention is formyl.

[0005] In addition, nocobactin NA in which the acyl group structure of the hydroxamic acid in the mycobactin acid moiety is acetyl (Biochemical Journal, Vol. 139, pp. 407-413, 1974), and BE-3230A classified into P-type mycobactin. , B, C, D and E (Journal of Antibiotics, Vol. 50, 815-
821, 1997), formobactin having a dimethyl structure in the cobactin moiety (Journal of Antibiotics 49: 839-8451, 1996),
Both mycobactins reported by Berkeley et al., Which are P-type (Journal of Bacteriology 164
896-903, 1985), mycobactin J (Current Microbiology 7, 337-341, 1982), and mycobactin S2 (Journal of American Chemical) having a structure in which the long fatty side chain of mycobactin S is substituted with a methyl group. Society 105 105 24
0-245 (1983)), but the compound of the present invention has a structure chemically different from any of these known mycobactin family compounds.

Mycobactin has iron chelating activity,
It is also known that the addition of the bacteria at the time of cultivation of the producing bacteria has a growth promoting effect (Bacteriological Reviews)
34, 99-125, 1970). Several other biological activities are known for mycobactin. For example, BE-
32030A, B, C, D and E have been reported to suppress the growth of cancer cells (Journal of Antibiotics 50: 815-821 1997).

[0007]

DISCLOSURE OF THE INVENTION A first object of the present invention is to provide a novel compound having excellent antitumor activity and antiviral activity, and useful as an antitumor or antiviral agent, and a method for producing the same. It is in. Further, a second object of the present invention is to provide an antitumor agent or an antiviral agent containing the compound as an active ingredient.

[0008]

Means for Solving the Problems The present invention provides (1) the following formula (I):

[0009]

Embedded image Or a pharmacologically acceptable salt thereof.

(2) A compound having the following physicochemical properties or a pharmacologically acceptable salt thereof: 1) Property: white powder; 2) [α] _D ²⁰ -7.1 ° (c1.0, MeOH); 3) Solubility: Soluble in dimethyl sulfoxide (DMSO), methanol, acetone, ethyl acetate. Insoluble in water; 4) Molecular formula: C ₄₀ H ₆₃ O ₁₀ N ₅ (measured by high-resolution mass spectrum); 5) Molecular weight: 773 (measured by FAB-MS spectrum); 6) Ultraviolet absorption spectrum: λmaxnm (ε) methanol The UV absorption spectrum measured in the solution is
243 (10600), 249 (10900), 258
(Sh, 5600), showing an absorption maximum at 304 (4700) nm; 7) Infrared absorption spectrum (KBr): The infrared absorption spectrum measured on a νmax cm ^-1 KBr disk is as follows: 3304, 2925, 2855, 173
9, 1665, 1642, 1534, 1492, 136
8, 1259, 1074, 756; 8) ¹ H-nuclear magnetic resonance spectrum; δ (ppm) in deuterated chloroform solution (tetramethylsilane (hereinafter “T
The nuclear magnetic resonance spectrum (360 MHz) measured on an internal standard) (referred to as "MS") is as follows: 0.88 (3H, t,
6.8 Hz), 1.17 (3H, d, 7.1 Hz), 1.2-2.1 (36 overlapping protons), 2.67 (1H, m), 3.46
(2H, t, 5.9 Hz), 3.74, (2H, m), 4.5-4.8 (4 overlapping protons), 4.96 (1H, t, 9.7 H
z), 4.99 (1H), 6.92 (1H, m), 7.03 (1H, d, 8.4 Hz),
7.19 (Exchangeable proton) (1H, br s), 7.21 (Exchangeable proton) (1H, br s), 7.43 (1H, m), 7.71 (1H, dd,
1.7 Hz, 7.9 Hz), 7.78 (1H, s) 9) ¹³ C-nuclear magnetic resonance spectrum; δ (ppm) Nuclear magnetic resonance spectrum (90 MHz) measured in deuterated chloroform solution (TMS internal standard) is as follows. Is as follows: 1
2.7 (q), 14.1 (q), 22.5 (t), 22.7 (t), 25.6 (t), 2
5.9 (t), 26.0 (t), 27.8 (t), 29.17 (t), 29.21 (t),
29.4 (t), 29.52 (t), 29.57 (t), 29.6-29.7 (5 overlapping signals) (t), 31.6 (t), 31.9
(t), 43.8 (d), 49.1 (t), 51.1 (d), 52.6 (t), 52.8
(d), 68.0 (d), 69.5 (t), 77.3 (d), 110.1 (s), 116.
9 (d), 119.3 (d), 128.7 (d), 134.3 (d), 156.8 (d),
159.6 (s), 167.9 (s), 169.0 (s), 170.9 (s), 171.2
(s), 171.4 (s) 10) High performance liquid chromatography Separation column: Symmetry ODS (φ4.6 × 150
mm, Waters) Mobile phase: 60% acetonitrile-30% methanol
10% water Flow rate: 1.0 ml / min Detection wavelength: 210 nm Retention time: 6.8 minutes (3) Culture the bacterium producing the compound according to (1) or (2) belonging to the genus Nocardia, A method for producing the compound according to (1) or (2), wherein the compound according to (1) or (2) is recovered from the culture; (4) a method belonging to the genus Nocardia (1) ) Or (2) is produced by Nocardia sp. SANK 62965.
(FERM BP-6179) strain, (5) Nocardia sp. (Nocardia sp.) SANK 62965 (FER
MBP-6179), (6) a medicament containing the compound according to (1) or (2) as an active ingredient, (7)
An apoptosis-inducing agent containing the compound according to (1) or (2) as an active ingredient, (8) (1) or (2)
An antitumor agent containing the compound as an active ingredient,
(9) An antiviral agent comprising the compound according to (1) or (2) as an active ingredient.

The present inventors have searched for substances having an activity of inhibiting the growth of WI38 / VA13 cells deficient in the p53 dysfunction of the tumor suppressor gene from among the secondary metabolites of microorganisms. As a result, they belong to the genus Nocardia isolated from natural soil. SANK 6
It has been found that a novel compound having excellent antitumor activity is produced in the culture solution of strain 2965 (FERM BP-6179). Furthermore, they have found that the novel compounds are also useful as antiviral agents, and have completed the present invention.

In the present invention, the compound described in the above (1) or (2) is referred to as "A-77543".

The compound A of the present invention represented by the above formula (I)
-77543 has some asymmetric carbon atoms and some double bonds. Therefore, there are various optical and geometric isomers. In the present invention, all of these isomers are represented by a single formula, but the present invention includes all of these isomers including racemates and mixtures of these isomers. When a stereospecific synthesis method is used, or when an optically active compound is used as a starting compound, individual isomers may be directly produced, while a mixture of isomers may be produced. The individual isomers may be obtained by a conventional method.

[0014] Compound A-77543 of the present invention can be salted using methods well known to those skilled in the art. The present invention also encompasses such salts of A-77543. A-775
The salt of No. 43 is not particularly limited as long as it is used medically and is pharmacologically acceptable. A-775
When the salt of 43 is used for a purpose other than medicine, for example, when it is used as an intermediate, there is no limitation. Such salts are preferably sodium salts, potassium salts,
Alkali metal salts such as lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts; Inorganic salts; t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N , N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt,
Amine salts such as organic salts such as piperazine salt, tetramethylammonium salt and tris (hydroxymethyl) aminomethane salt; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt and asparagine salt. Can be. More preferably, those preferably used as pharmacologically acceptable salts, that is, sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt and the like can be mentioned.

The compound of the present invention or a salt thereof may be a solvate. For example, by leaving it in the air or by recrystallizing, it absorbs moisture,
The solvate may be adsorbed water or form a hydrate, and such a solvate is also included in the present invention.

The present invention further includes all compounds which are metabolized in the living body and converted into A-77543, so-called prodrugs.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION As a strain belonging to the genus Nocardia used in the production of compound A-77543 of the present invention, there can be mentioned, for example, Nocardia sp. SANK 62965 strain. SANK 62965 strain was isolated from a diluent obtained by the soil dilution method from soil collected in Nagoya City, Aichi Prefecture, and its mycological characteristics are as described below.

1. Morphological characteristics SANK 62965 strain was obtained from ISP [International Streptomyces Project (Internationa
l Streptomyces Project)] on a defined agar medium at 28 ° C,
After culturing for 14 days, SANK 629 was observed under a microscope.
The 65 basic mycelia elongate and diverge favorably, exhibiting a yellowish gray to light olive color, but no hypha rupture or zigzag elongation like Nocardia strains is observed. Aerial hyphae are simply branched. Ten to fifty or more spore chains are formed at the tips of the aerial hyphae, and the spore chain forms a loose spiral. In observation with a scanning electron microscope,
The surface structure of the spores is spiny. The spores are elliptical and have a size of 0.6 to 0.8 x 0.8 to 1.
2 μm. In addition, no special organs such as axle branching of aerial hyphae, sclerotium and sporangia are observed.

2. Properties on various culture media The properties after cultivation on various culture media at 28 ° C for 14 days are shown in Table 1 below.
As shown in FIG. The display of the color tone indicates the color chip number of the "Standard Color Chart" of the Japan Color Research Institute using the Munsell method.

[0020]

[Table 1] Type of medium Item ^{* 1} Properties of SANK 62965 strain Sucrose G: Not good, flat, yellowish ash (5Y 9/1) ^{* 2} Nitrate agar AM: Good, velvety, bright brownish ash (10YR) 7/2) R: brownish white (2.5Y 9/1) SP: not produced glucose G: good, flat, brownish white (2.5Y 9/1) asparagine agar AM: not very good, velvety, white R : Yellowish ash (5Y 9/1) SP: Not producing glycerin G: Good, flat, yellowish ash (5Y 9/1) Asparagine agar AM: Not very good, velvety, white (ISP5) R: Yellow Taste ash (7.5Y 9/2) SP: Not produced, starch / inorganic salt agar G: Good, flat, yellow ash (5Y 9/1) (ISP5) AM: Abundantly formed, velvety, tea ash (10YR) 6/2) R: Kimihai (7.5Y 9/3) SP: tyrosine agar not produce G: not so good, flat, Kimihai (5Y 9/1) (ISP7) AM : very well There, velvety, white R: Kimihai (7.5Y 9/2) SP: nutrient agar not produce G: very good, flat, Kimihai (5Y 9/2) (Difco) AM : abundantly formed Velvet, white to light brown (2.5Y 8/2) R: light yellow (5Y 9/3) SP: no yeast extract G: very good, flat, light olive (5Y 8/3) malt extract Agar AM: Abundantly formed, fluffy, pale yellowish tea (2.5Y 7/2) (ISP2) R: pale yellow (5Y 9/6) SP: oatmeal agar without producing G: very good, flat, yellowish ash (5Y 8/2) (ISP3) AM: Abundantly formed, velvety, light brownish ash (10YR 7/2) R: Yellowish ash (7.5Y 9/3) SP: No production, water agar G: Very good No, flat, yellowish ash (5Y 9/1) AM: good, velvety, light brown (2.5Y 8/2) R: yellowish ash (5Y 9/1) SP: no potato extract, G: good No, flat, yellowish ash (5Y 9/1) Ginseng extract agar A : Good, velvety, light brown (2.5Y 8/2) R: Kimihai (5Y 9/1) SP: do not produce * 1 G: growth, AM: aerial mycelium, R: back, SP: soluble pigment * 2. The color in parentheses in the property column is the color tone display by the Munsell method. Physiological properties SANK observed for 2 to 21 days after incubation at 28 ° C
The physiological properties of the 62965 strain are as shown in Table 2.

[0021] * Medium 1: Tryptone yeast extract broth (IS
P1), medium 2: peptone / yeast extract / iron agar (ISP6), medium 3: tyrosine agar (ISP7), medium 4: yeast extract / malt extract agar (ISP2).

Table 3 shows the assimilation of the carbon source of the SANK 62965 strain observed after cultivation at 28 ° C. for 14 days using Prideham Gottlieb agar medium (ISP9).
As shown in FIG.

[0023]

[Table 3] Carbon source assimilation D-glucose + D-fructose ± D-arabinose-L-rhamnose-D-xylose-sucrose-inositol + raffinose-D-mannitol + control- +: used, ±: weak Use,-: Do not use. 4. About the cell components The cell wall of the SANK 62965 strain was prepared by the method of Becker, B. et al., (1984) Applied Microbiolog.
y 12, 421-423), the detection of L, L-diaminopimelic acid confirmed the cell wall type I. Moreover, the sugar component in all the cells of the SANK 62965 strain was determined by the method of Lechevalier (Lechevalier,
MP (1968) Journal of Laboratory & Clinical Med
icine 71, 834), no characteristic pattern was observed.

ISP [International Streptomyces Project (International Streptomyces Project)
Project)], by Waxman, SA Waksman, The Actinomycetes, Volume 2, Buchanan and Gibbons, Barges Manual (R.
E. Buchanan and NE Gibbons, Bergey's Manual of
Determinative Bacteriology, 8th edition (1974), Bergey's Manual of Systemati
c Bacteriology), Volume 4 (1989), and recent literature on actinomycetes of the genus Nocardia.
rdia). Therefore, this strain was transferred to Nocardia sp.
The strain was named 5 strains. This strain was obtained on November 21, 1997.
It is internationally deposited with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology, under the deposit number FERM BP-6179.

As is well known, actinomycetes can be used in nature or by artificial manipulation (for example, ultraviolet irradiation, irradiation,
Mutation easily occurs due to chemical treatment, etc., and this is the same for the SANK 62965 strain of the present invention. The SANK 62965 strain referred to in the present invention includes all mutant strains.

[0026] These mutants also include those obtained by genetic methods, for example, recombination, transduction, transformation and the like. That is, the SANK 62965 strain and its mutant strains that produce A-77543, and strains that are not clearly distinguished therefrom, are all SANK.
62965 strains.

5. For culturing To obtain A-77543, the culturing of the microorganism is performed in a medium such as that used to produce other fermentation products. Such a medium contains a carbon source, a nitrogen source and inorganic salts that can be assimilated by the microorganism.

Generally, glucose, fructose, maltose, sucrose, mannitol,
Glycerin, dextrin, oats, rye, corn starch, potato, corn flour, cottonseed oil, molasses, citric acid, tartaric acid and the like can be used alone or in combination. In general, the amount is varied from 1 to 10% by weight of the medium volume.

As the nitrogen source, a substance containing protein and its hydrolyzate or an inorganic salt is generally used in the fermentation step. Suitable nitrogen sources include soybean flour, bran, peanut flour, cottonseed flour, casein hydrolyzate, pharmamine, fish meal, corn steep liquor, peptone, meat extract, yeast, yeast extract, malt extract, sodium nitrate, ammonium nitrate , Ammonium sulfate and the like. The nitrogen source is used alone or in combination in the range of 0.2 to 10% by weight of the medium volume.

The nutrient inorganic salts to be taken into the medium are ordinary salts from which ions such as sodium, ammonium, calcium, phosphate, sulfate, chloride and carbonate can be obtained. It also contains trace metals such as potassium, calcium, cobalt, manganese and magnesium.

In the liquid culture, silicone oil, vegetable oil, surfactant and the like are used as antifoaming agents. SAN
The pH of the medium for culturing the K62965 strain and producing A-77543 can be changed to 5.0 to 8.0.

The strain grows in the range of 14 to 35 ° C.
Particularly, it is good in the range of 21 ° C to 30 ° C. A-
A temperature of 22 ° C. to 28 ° C. is suitable for the production of 77543.
A-77543 is obtained by aerobic culturing, and aerobic culturing methods usually used, for example, a solid culturing method, a shaking culturing method, an aeration stirring culturing method and the like are used.

In small-scale culture, it is preferable to carry out shaking culture at 28 ° C. for several days. The culture is initiated by one or more stages of seed development in an Erlenmeyer flask. The seed flask is shaken in a constant temperature incubator for several days, or until fully grown. The grown seed is used to inoculate part or all of the seed in the next stage seed or production medium. The flask containing the inoculated production medium is shaken at a constant temperature for several days, and after completion of the culture, the contents of the flask are separated by centrifugation or filtration. In the case of mass culture, it is preferable to culture in a suitable tank equipped with a stirrer and aeration device. According to this method, a nutrient medium can be prepared in a tank. The nutrient medium is sterilized by heating to 121 ° C., and after cooling, the sterile medium is inoculated with seeds that have been grown in advance. Culture is performed at 28 ° C. with aeration and stirring. This method is suitable for obtaining large amounts of compounds.

A-775 produced as the culture progresses
To know the time-dependent change in the amount of 43, for example, the filtrate obtained by filtering the culture was used to filter the WI38 /
It is measured by performing a growth inhibition test on VA13 cells. Normally, the production of A-77543 reaches the maximum value after 96 to 180 hours of culture.

6. After completion of the extraction / purification culture, A-77543 present in the culture solution is separated from the bacterial cells and other solid parts by a filtration operation using diatomaceous earth as a filter aid or by centrifugation, and is present in the filtrate or the bacterial cells. A-7754
3 can be obtained by extraction and purification utilizing its physicochemical properties.

For example, activated carbon or a resin for adsorption, such as Amberlite XAD-2 and XAD-4 (both manufactured by Rohm and Haas), Diaion HP-10 and HYDON
P-20, HP-50, CHP20P (all manufactured by Mitsubishi Chemical Corporation) and the like are used. The liquid containing A-77543 is passed through the adsorbent layer as described above to adsorb and remove impurities, or after adsorbing A-77543, a mixture of water such as methanol water or acetone water and an organic solvent. It is obtained by eluting with a solvent.

The thus obtained A-77543
Are further separated by adsorption column chromatography using a carrier such as silica gel or florisil, distribution column chromatography using Avicel (manufactured by Asahi Kasei Corporation), Sephadex LH-20 (manufactured by Pharmacia), and normal phase. And high-performance liquid chromatography using a reversed-phase column.

Further, the solution containing A-77543 is mixed with an ion-exchange resin, for example, an organic solvent immiscible with water, for example,
Purification by extracting and removing impurities with n-butanol, ethyl acetate, methylene chloride or the like alone or in combination thereof is also possible.

The location of the compound A-77543 of the present invention in the purification can be determined by the following methods.

1) Growth Inhibition Test of Tumor Suppressor Gene p53 Deficient Cell: Normal cell line and tumor suppressor gene p53 dysfunctional cell line of the cell line (for example, human normal cell line WI
38 and its p53 dysfunctional cell line VA-13) are each cultured for a certain period of time in the presence of a test sample. afterwards,
The number of living cells or a value reflecting the number of living cells is measured by a method known to those skilled in the art, such as an XTT test, and the effects of the test sample on cell growth are compared. A-77543 selectively inhibits the growth of p53 dysfunctional cell lines at low concentrations.

2) Apoptosis induction test on p53 dysfunctional cells: A normal cell line and p
After culturing each of the 53 dysfunctional cell lines in the presence of a test sample for a certain period of time, it was determined whether apoptosis was induced in each cell line by using, for example, an ELISA kit for apoptosis quantification using chromatin fragmentation as an index. Investigate using. A-77543 selectively induces apoptosis in p53 dysfunctional cell lines at low concentrations.

3) Tumor growth inhibitory action in vivo: A tumor cell (for example, human colon cancer strain Col-2-JCK) is implanted subcutaneously in the axillary region of a nude mouse, and a test sample is periodically intraperitoneally administered. While the animals are kept for a certain period of time, the increase in the volume of the transplanted tumor is measured. A-77543 also inhibits tumor growth in vivo.

4) Effect on production of human immunodeficiency virus (HIV): A human cell line infected with HIV is cultured for a certain period of time in the presence of a test sample, and H produced during the culture.
The IV amount is measured. A-77543 suppresses HIV production in a concentration-dependent manner.

The compound A-77543 of the present invention is useful as an antitumor agent. A-77543 is HIV
-1 is also useful as an antiviral agent, especially an anti-HIV agent, because it also inhibits the growth of virus in persistently infected cells in a concentration-dependent manner.

When the compound of the present invention is used as an antitumor agent or an antiviral agent, it is administered in various forms. Examples of the administration form include oral administration by tablets, capsules, powders, granules, syrups and the like. Or injections (intravenous, intramuscular, subcutaneous) and the like can be mentioned. These various preparations are commonly used in the field of pharmaceutical preparations such as excipients, binders, disintegrants, lubricants, flavoring agents, dissolution aids, dissolution aids, suspensions, coatings, etc. in the main drug in accordance with ordinary methods. It can be formulated using known adjuvants.

For forming into tablets, those conventionally known in the art can be widely used as a simple substance, such as lactose, sucrose, sodium chloride, glucose, urea, starch,
Excipients such as calcium carbonate, kaolin, crystalline cellulose, silicic acid, etc .; binding of water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc. Ingredients: dried starch, sodium alginate, agar powder, laminaran powder,
Disintegrators for sodium hydrogencarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, disintegrators for sucrose, stearin, cocoa butter, hydrogenated oil, etc .; Absorption promoters such as ammonium base and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; purified talc, stearate, boric acid powder, polyethylene glycol, etc. And the like. Further tablets are tablets coated with normal skin as needed,
For example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double tablets or multilayer tablets can be prepared.

In the case of molding into pills, carriers conventionally known in the art can be widely used as carriers, for example, excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, and talc; Binders such as rubber powder, tragacanth powder, gelatin, and ethanol; disintegrators such as laminaran agar;

For shaping in the form of suppositories, those conventionally known in the art can be widely used as carriers, for example, polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. Can be mentioned.

When prepared as an injection, the liquid and suspensions are preferably sterilized and isotonic with blood. When formed into the form of these solutions, emulsions and suspensions, All agents commonly used in this field can be used, for example, water, ethanol, propylene glycol, ethoxylated isostearyl alcohol,
Examples thereof include polyoxylated isostearyl alcohol and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of salt, glucose, or glycerin to prepare an isotonic solution may be included in the pharmaceutical preparation, and ordinary solubilizers, buffers, soothing agents, etc. May be added.

Further, a coloring agent, a preservative, a flavor, a flavoring agent, a sweetening agent and the like and other pharmaceuticals may be contained, if necessary.

The amount of the active ingredient compound contained in the above-mentioned pharmaceutical preparation is not particularly limited and may be appropriately selected in a wide range, but is usually 1 to 70% by weight, preferably 1 to 30% by weight of the whole composition.
It is appropriate that the amount is contained by weight%.

The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. In the case of an injection, it may be administered intravenously, alone or as a mixture with a normal replenisher such as glucose or amino acid, and, if necessary, intramuscularly, intradermally, subcutaneously or intraperitoneally. In the case of suppositories, they are administered rectally.

The amount used depends on symptoms, age, body weight, administration method, dosage form and the like.
It is preferable to administer 1 mg to 1000 mg per day in one or several divided doses.

[0054]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited thereto.

Embodiment 1 Preparation of A-77543 (A) Culture Nocardia SP. (Nocardias
p. ) SANK 62965 strain (FERM BP-61)
79) was inoculated from a slant into two 500 ml Erlenmeyer flasks each containing 100 ml of the medium represented by Medium Composition-1 and was subjected to rotation at 210 rpm and 2 rpm using a rotary shaking incubator.
What was cultured at 8 ° C. for 168 hours was used as a seed culture solution. Next, 100 ml of the medium represented by Medium Composition-2 was added to 500 m
The mixture was placed in 30 1-L Erlenmeyer flasks and sterilized by heating at 121 ° C. for 45 minutes. After cooling this to 28 ° C., 2 m of seed culture
1 and incubated at 210 rpm at 28 ° C. for 168 hours with stirring.

[Medium composition-1] Dextrin 6% Glucose 1% Oatmeal 2% Soybean flour 1.0% Cobalt chloride 0.0002% Calcium carbonate 0.5% Sodium chloride 0.1% Dipotassium phosphate 0.1% Magnesium sulfate heptahydrate 0.3% Antifoam CB-442 0.01% pH 7.0 before sterilization.

[Medium composition-2] Glucose 5.0% Pharmamedia 0.6% Soybean powder 0.75% Dipotassium phosphate 0.1% Calcium carbonate 0.55% Ammonium sulfate 0.5% Defoamer CB- 442 0.01% pH 7.0 before sterilization.

(B) Isolation Celite 5 was added to 3 liters of the obtained culture solution as a filter aid.
To the mixture was added 150 g of 45 (manufactured by Johns Manville Product Corporation), followed by filtration to obtain a cell cake. 1.5 L of 80% acetone water was added thereto, and the mixture was extracted with stirring for 1 hour, followed by filtration to obtain an acetone extract. Concentrate 1500 ml of the acetone extract under reduced pressure
ml were obtained. The pH of 500 ml of the concentrated solution was adjusted to 4.0, and extracted twice with an equal volume of ethyl acetate. 0.2 ml of extract
M, washed successively with an aqueous solution of disodium phosphate and saturated saline, dehydrated with anhydrous sodium sulfate, filtered with a filter paper, concentrated to dryness under reduced pressure, and dried to obtain 1.12 g of an extract containing A-77543.
I got Next, 1.12 g of this powder was separated and purified using HPLC. HPLC column (YMCO
DS (15/30) 100 × 500 mm) was equilibrated with 60% acetonitrile-30% methanol-10% water,
Dissolve 1.12 g of the extract powder in 100 ml of the same solvent, inject the whole amount as a sample solution, and then add 60% acetonitrile-30.
% Methanol-10% water as a solvent at a flow rate of 190 ml /
Eluted in minutes. Monitor eluate at detection wavelength of 215nm
The eluate having a retention time of 46 to 53 minutes was collected, and the fractionated solution was concentrated to dryness to obtain 400 mg of A-77543 white powder.

A part of this white powder was collected and analyzed by high performance liquid chromatography to obtain the following results: Separation column: Symmetric ODS (φ4.6 × 150 m)
mobile phase: 60% acetonitrile-30% methanol-
10% water; flow rate: 1.0 ml / min; temperature: 25 ° C; detection wavelength: 210 nm; retention time: 6.8 minutes.

Test Example 1 Growth inhibition test of WI38 / VA13 cells with tumor suppressor gene p53 dysfunction VA13 cells (American Type Culture Collection (hereinafter referred to as "ATCC") CCL-75.
1) and WI38 cells (ATCC CCL-75) were used. MEM (manufactured by Gibco) containing 10% fetal bovine serum (manufactured by HiClone) was used as a culture solution. First, VA1
3 cells (1500 cells / well) or (7600 cells / well) were transferred to a 96-well flat bottom plate (Corning
3598) (Costar), 5% CO ₂ , 37 ° C
The cells were cultured under the conditions for 2-3 days. The compound obtained in Example 1 was diluted with a medium in advance to a final volume of 200.
The mixture was added at −220 μl and cultured at 37 ° C. for 24 hours. Thereafter, the medium was removed and the fresh medium was
1 / well was added and the cells were further cultured at 37 ° C. for 72 hours.
After completion of the culture, an XTT / PMS solution (1 mg / ml 2,3
-Bis [2-methoxy-4-nitro-5-sulfophenyl] -2H-tetrazolium-5-carboxanilide (manufactured by Sigma) and 25 mM phenazine / methosulfate (manufactured by Sigma) dissolved in a culture solution (50 μl)
1 / well, incubate at 37 ° C. for 2-4 hours, plate reader (Spectra Max 25)
0, manufactured by Molecular Devices, Inc.) at 450 nm. The incubation time was used as a guide when the absorbance of the wells to which no sample was added was 1 to 1.5. The percentage of viable cells (%) was calculated based on the following formula: Percentage of viable cells (%) = (absorbance of sample-added / cell-added well−absorbance of cell-unadded well) × 100 / (sample-unadded / cell-added well) Absorbance-absorbance of wells without cells).

Finally, the log of the concentration and the percentage of viable cells are
Create a plotted graph to reduce live cell percentage by 50%
Concentration (hereinafter referred to as “IC₅₀Value))
It was an index of sex. A-77543 is a function of VA13 cells.
0.013 μM (0.028 μg / ml) IC₅₀value
Was toxic, whereas IC against WI38 cells was ₅₀
The value is about 19 μM (40 μg / ml), 1000 times
The above differences were observed. In addition, culture time before drug addition
(2-3 days) by IC₅₀Is there a significant difference between the values?
Was.

Test Example 2 Apoptosis Induction Test for Tumor Suppressor Gene p53-Deficient Cells An ELISA kit (Cell Death Detection ELISA)
A, manufactured by Boehringer Mannheim). First, cells were placed in a 96-well flat bottom plate (Corning Costar 3598) at 200 μl / well (WI3
8 for 16000 cells / well or VA13 for 4000
Cells / well), and cultured for 24 hours. Sample 2 there
0 μl was added, and the cells were further cultured for 24 hours. Thereafter, each well was washed twice with 200 μl / well of a phosphate buffered saline (hereinafter referred to as “PBS”, manufactured by Nissui Pharmaceutical Co., Ltd.) except the medium. After removing the washing solution, a cell lysis buffer (bottle 5 attached to the kit) was added at 200 μl / well, and the mixture was stirred with a plate mixer (Micromixer E-36, manufactured by Taitec Co., Ltd.). Plate 200
After centrifugation at × g for 5 minutes to precipitate high molecular DNA,
20 μl of the supernatant from each well was transferred to a streptavidin-coated plate. Further, a mixed solution of a biotin-labeled anti-histone antibody and a peroxidase-labeled anti-DNA antibody (bottle 2) was added at 80 μl / well, and the mixture was stirred at room temperature for 2 hours.
Incubated for hours. After the reaction, the antibody solution was removed and 25
Washed three times with 0 μl / well incubation buffer (bottle 4). After removing the liquid, 2,2′-azinobis (3-ethylbenzthiazolinesulfonic acid (AB
TS) A substrate solution (bottles 6 and 7) was added at 100 μl / well, color was developed at room temperature, and the absorbance at 405 nm (OD405) was measured with a plate reader.

[0063]

Table 4 Apoptosis induction on p53 dysfunctional cells A-77543 DNA fragmentation (OD405, mean ± standard deviation) (μM) WI38 VA13 1.3 × 10 ² 0.132 ± 0.008 0.646 ± 0.248 1.3 × 10 ⁰ 0.002 ± 0.005 0.338 ± 0.010 1.3 × 10 ^{− 1} 0.002 ± 0.005 0.275 ± 0.068 1.3 × 10 ^-2 0.004 ± 0.008 -0.001 ± 0.031 1.3 × 10 ^-3 -0.001 ± 0.004 -0.021 ± 0.017 1.3 × 10 ^-4 -0.003 ± 0.002 -0.015 ± 0.023 1.3 × 10 ^-5 -0.009 ± 0.007 -0.045 ± 0.016 1.3 × 10 ^-7 -0.003 ± 0.008 -0.003 ± 0.019 Test Example 3. Tumor growth inhibitory effect on human colon cancer strain Col-2-JCK cells The compound A-77543 of the present invention was produced in vivo.
o) has a tumor growth inhibitory effect. This growth inhibitory effect was determined from the increase and decrease in tumor volume.

First, an approximately 3 mm square human colon cancer strain Col-2-JCK tumor piece was subcutaneously subcutaneously subcutaneously in the axillae of six Balb / c nude mice (female, 8 weeks old) in a group of 6 mice (3.5 mm inner diameter). Was transplanted using. A obtained in Example 1
-77543 is suspended in sterile distilled water containing 5% dimethylacetamide (DMA) and 5% polyoxyethylene hydrogenated castor oil (HCO-60), and is 1.6 mg / kg and 6.3 mg / kg once a day.
Was administered intraperitoneally three times on days 7, 11 and 15 after tumor piece implantation.

The tumor growth inhibitory effect on human colon cancer strain Col-2-JCK was evaluated by calculating the tumor growth inhibition rate (TGI (%)) on the 25th day after tumor implantation from the following formula.
That is, the relative tumor volume ratio (RTV) of each mouse on day 25 after tumor implantation was first calculated according to the following equation,
After averaging 6 animals, the tumor growth inhibition rate (TGI (%)) of the compound-administered group relative to the untreated control group was calculated: Relative tumor volume ratio (RTV) = Tumor volume 25 days after tumor implantation ^* / Tumor volume on day 7 after tumor piece transplantation ^* Tumor growth inhibition rate (TGI) (%) = (1-RTVt / R)
TVc) RTVt = Relative tumor volume ratio of compound administration group RTVc = Relative tumor volume ratio of untreated control group * Tumor volume = 1/2 x (tumor major axis) x (tumor minor axis) ^{2 The} results are shown in Table 5.

[0066]

[Table 5] The compound A-77543 of the present invention is a human colorectal cancer strain Col
-2- Proliferation of JCK was suppressed in vivo.

Test Example 4 Effect on HIV production HIV-1IIIB persistently infected MOLT in logarithmic growth phase
-4 cells (MOLT-4 / IIIB cells)
It was collected in a sedimentation tube and centrifuged at 200 × g for 3 minutes. Up
After discarding the supernatant, the remaining cell pellet is suspended in PBS and re-suspended.
And 200 × g for 5 minutes. This centrifuge and PBS
After repeating the operation of suspension twice, 10% fetal bovine blood
Suspended in a clear containing RPMI1640 medium (manufactured by Gibco),
1 × 10 ⁶A cell / ml cell suspension was prepared. This fine
The cell suspension was previously prepared with various concentrations of A-77543.
RPMI 1640 medium containing 0% fetal calf serum
l / well 96-well plate
100 μl / well in 3096)
Each time (n = 3), and add 5% CO 2 to the plate._Two, 3
The cells were cultured at 7 ° C. for 24 hours. After the culture is completed,
The concentration of HIV p24 antigen is determined by HIV p24 antigen ELI.
Quantification using SA kit (manufactured by Cellular Products)
did. Average p24 antigen concentration in wells without A-77543
The average when each concentration of A-77543 was added, with the degree as 100%.
The p24 concentration and the standard deviation were calculated, and the HIV production index was calculated.
Mark. Also, remove the culture supernatant by 50 μl / well.
Thereafter, 50 μl / well of XTT / PMS solution was added,
The percentage of live cells was determined.

As a result, it was revealed that A-77543 suppressed HIV production at a concentration that did not decrease the percentage of living cells (FIG. 1).

[0069]

As described above, the novel compound A-7 which has excellent antitumor activity and antiviral activity according to the present invention and is useful as a medicine, especially as an antitumor agent or antiviral agent
7543, and a method for producing the same, and an antitumor agent or an antiviral agent containing the compound as an active ingredient. The malignant tumor that can be treated by administering a drug containing the compound of the present invention as an active ingredient is not particularly limited, and includes, for example, gastric cancer, esophageal cancer, pancreatic cancer,
Liver cancer, colon cancer, colon cancer, rectal cancer, cervical cancer, uterine body cancer, ovarian cancer, breast cancer, skin cancer, prostate cancer, testicular tumor, head and neck cancer, brain tumor, leukemia, malignant lymphoma, bladder cancer, etc. Can be

[Brief description of the drawings]

FIG. 1 shows the effect of A-77543 on HIV production.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12R 1:01) (72) Inventor Shinichi Kurata 1-258 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. Inside the formula company (72) Inventor Hideyuki Shiozawa 1-2-58 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Mutsuo Nakajima 1-2-58 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Hidehiko Furukawa 1-258 Hiromachi, Shinagawa-ku, Tokyo F-term (reference) 4B064 AE54 BA08 BE09 BE12 BE14 BE19 BG01 BG09 BH01 BH02 BH04 BH05 BH06 BH07 CA03 DA02 DA05 4B065 AA38X AC14 BA22 CA18 CA44 4C063 AA01 BB07 CC52 DD19 EE01

Claims (9)

[Claims] (1) The following formula (I): Or a pharmacologically acceptable salt thereof. 2. A compound having the following physicochemical properties or a pharmacologically acceptable salt thereof: 1) Property: white powder; 2) [α] _D ²⁰ −7.1 ° (c1.0, MeOH) 3) Solubility: Soluble in dimethyl sulfoxide (DMSO), methanol, acetone and ethyl acetate. Insoluble in water; 4) Molecular formula: C ₄₀ H ₆₃ O ₁₀ N ₅ (measured by high-resolution mass spectrum); 5) Molecular weight: 773 (measured by FAB-MS spectrum); 6) Ultraviolet absorption spectrum: λmaxnm (ε) methanol The UV absorption spectrum measured in the solution is
243 (10600), 249 (10900), 258
(Sh, 5600), showing an absorption maximum at 304 (4700) nm; 7) Infrared absorption spectrum (KBr): The infrared absorption spectrum measured on a νmax cm ^-1 KBr disk is as follows: 3304, 2925, 2855, 173
9, 1665, 1642, 1534, 1492, 136
8, 1259, 1074, 756; 8) ¹ H-nuclear magnetic resonance spectrum; δ (ppm) in deuterated chloroform solution (tetramethylsilane (hereinafter “T
The nuclear magnetic resonance spectrum (360 MHz) measured on an internal standard) (referred to as "MS") is as follows: 0.88 (3H, t,
6.8 Hz), 1.17 (3H, d, 7.1 Hz), 1.2-2.1 (36 overlapping protons), 2.67 (1H, m), 3.46
(2H, t, 5.9 Hz), 3.74, (2H, m), 4.5-4.8 (4 overlapping protons), 4.96 (1H, t, 9.7 H
z), 4.99 (1H), 6.92 (1H, m), 7.03 (1H, d, 8.4 Hz),
7.19 (Exchangeable proton) (1H, br s), 7.21 (Exchangeable proton) (1H, br s), 7.43 (1H, m), 7.71 (1H, dd,
1.7 Hz, 7.9 Hz), 7.78 (1H, s) 9) ¹³ C-nuclear magnetic resonance spectrum; δ (ppm) Nuclear magnetic resonance spectrum (90 MHz) measured in deuterated chloroform solution (TMS internal standard) is as follows. Is as follows: 1
2.7 (q), 14.1 (q), 22.5 (t), 22.7 (t), 25.6 (t), 2
5.9 (t), 26.0 (t), 27.8 (t), 29.17 (t), 29.21 (t),
29.4 (t), 29.52 (t), 29.57 (t), 29.6-29.7 (5 overlapping signals) (t), 31.6 (t), 31.9
(t), 43.8 (d), 49.1 (t), 51.1 (d), 52.6 (t), 52.8
(d), 68.0 (d), 69.5 (t), 77.3 (d), 110.1 (s), 116.
9 (d), 119.3 (d), 128.7 (d), 134.3 (d), 156.8 (d),
159.6 (s), 167.9 (s), 169.0 (s), 170.9 (s), 171.2
(s), 171.4 (s) 10) High performance liquid chromatography Separation column: Symmetry ODS (φ4.6 × 150
mm, Waters) Mobile phase: 60% acetonitrile-30% methanol
10% water Flow rate: 1.0 ml / min Detection wavelength: 210 nm Retention time: 6.8 minutes. 3. The method according to claim 1, wherein a bacterium producing the compound according to claim 1 or 2 belonging to the genus Nocardia is cultured, and the compound according to claim 1 or 2 is recovered from the culture. Item 10. The method for producing a compound according to item 1 or 2. 4. The bacterium according to claim 1 or 2, which belongs to the genus Nocardia.
Espy (Nocardia sp.) SANK 62965 (FE
RM BP-6179) strain. 5. Nocardia Espy
p.) SANK 62965 (FERM BP-617)
9). 6. A pharmaceutical comprising the compound according to claim 1 as an active ingredient. 7. An apoptosis-inducing agent comprising the compound according to claim 1 or 2 as an active ingredient. 8. An antitumor agent comprising the compound according to claim 1 as an active ingredient. 9. An antiviral agent comprising the compound according to claim 1 as an active ingredient.